In all eukaryotic cells, a central and common process exists for integral membrane protein and secretory protein post-translational modification and delivery. This common process involves vesicular transport from their site of synthesis, the lumen of the rough endoplasmic reticulum (ER), to and through the Golgi apparatus, and ultimately to specific plasma membrane or internal membrane domains. See, e.g., Darnell et al., Molecular Cell Biology (1990, Scientific American Books) at Chapter 17, "Plasma-Membrane, Secretory, and Lysosome Proteins: Biosynthesis and Sorting."
Following protein synthesis in the ER, most proteins travel via small transport vesicles to the Golgi complex, an organelle composed of both flattened and spherical vesicles that serve as a liaison between the ER and both the plasma membrane and internal organelles, such as lysosomes. The Golgi complex contains three functional regions: (1) the elongated vesicles nearest the ER make up the cis face of the Golgi (2) those of the mid-portion, the medial face; and (3) those nearest the periphery of the cell, the trans face and the trans Golgi reticulum. Glycosylation is the principal chemical modification that occurs to proteins as they pass through the Golgi apparatus. See, e.g., Darnell et al., Molecular Cell Biology (1990, Scientific American Books) at Chapter 17, "Plasma-Membrane, Secretory, and Lysosome Proteins: Biosynthesis and Sorting."
The ankyrins are a family of proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. Ankyrins are known to link the spectrin cytoskeleton and integral membrane proteins. Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton, Devarajan et al., PNAS: 2965-69 (1994). Multiple isoforms of ankyrin have arisen both by gene duplication as well as by alternative transcription, Devarajan et al., PNAS 91: 2965-69 (1994).
Spectrins and actin are the major components of a cell's cortical or membrane cytoskeleton. The membrane cytoskeleton is the major determinant of the rigidity of cell membranes and acts to restrict the lateral motion of membrane glycoproteins. The major constituents of the cytoskeleton are .alpha.- and .beta.-spectrin and actin. Spectrin binds to the sides of actin microfilaments producing the lace-like cytoskeleton. Darnell et al., Molecular Cell Biology (1990, Scientific American Books) at Chapter 13, "The Plasma Membrane."
A. Ankyrins
Interactions between integral membrane proteins and the underlying spectrin-actin cytoskeleton play key roles in such activities as cell motility, activation, proliferation, contact, and the maintenance of specialized membrane domains (Luna and Hitt, Science 258: 955-64 (1992); Bennett and Gilligan, Annu. Rev. Cell Biol. 9: 27-66 (1993); Devarajan and Morrow, Membrane Protein-Cytoskeleton Complexes: Protein Interactions, Distributions and Functions (1996, Academic Press); Morrow et al., Handbook of Physiology (1997, Oxford Press). Ankyrins are a family of large membrane associated proteins that have emerged as crucial adapter molecules mediating such linkages, since they possess recognition sites for various membrane proteins as well as for cytoskeletal elements, Bennett, J. Biol. Chem. 267: 8703-6 (1992). Molecules employing a 33 residue repetitive structure first identified in the 89 kDa domain I of ankyrin display a wide tissue distribution. Bork, Proteins 17: 363-74 (1993); Chan et al., J. Cell Biol. 123: 1463-73 (1993); Axton et al., EMBO. J. 13: 462-70 (1994); Diederich et al., Develop. 120: 473-81 (1994), and tissue-specific isoforms may be present in all cells (Lux et al., Nature 344: 36-42 (1990a).
Several distinct isoforms of ankyrin have been recognized by their immunological properties. The isoform associated with the membranes of red cells, neuronal cell bodies and dendrites has been termed Ank.sub.R. Antibodies to Ank.sub.R also cross-react with an ankyrin found in the basolateral membrane of polarized epithelial cells. Davis et al., J. Biol., Chem. 264: 6417-26 (1989); Morrow et al., J. Cell Biol. 108: 455-65 (1989)). Ank.sub.R is encoded by the ANK1 gene (Lux et al., (1990a); Lux et al., Nature 345: 736-39 (1990b)), which transcribes mRNA species of 7 kilobases (Kb) in erythrocytes (Lambert et al., 1990; Lux et al., Nature 344: 36-42 (1990a)) and 9 Kb in brain (Lambert et al., 1990).
Ank.sub.B is the major isoform in brain, and is widely present in neuronal and glial cell membranes, as well as in a variety of non-neuronal tissues including kidney. It is the product of the ANK2 gene (Otto et al., J. Cell Biol. 114: 241-53 (1991)). At least two alternatively spliced mRNA transcripts of 9 Kb and 13 Kb are generated from this gene (Kunimoto et al., J. Cell Biol. 115: 1319-31 (1991); Otto, J. Cell Biol. 114: 241-53 (1991)).
Ank.sub.G is an immunologically distinct isoform found at the plasma membrane at the nodes of Ranvier and at the axon initial segments (Kordeli et al., J. Cell Biol. 110: 1341-52 (1990); Kordeli and Bennett, J. Cell Biol. 114: 1243-59 (1991); Kordeli et al., J. Biol. Chem. 270: 2352-9 (1995)). It is the product of the ANK3 gene (Peters et al., J. Cell Biol. 130: 313-30 (1995)). Although two transcripts of the Ank.sub.G gene (15 Kb and 10 Kb) are neural-specific, smaller alternatively spliced isoforms may be expressed in kidney and lung (Kordeli et al., J. Biol. Chem. 270: 2352-9 (1995); Peters et al., J. Cell Biol. 130: 313-30 (1995)). In addition, a truncated ankyrin of 72 kilo-Daltons (kDa), which binds spectrin and is localized with the membrane protein GP85, has been detected immunologically in T-lymphocytes; its gene of origin is unknown (Bourguignon et al., J. Cell Biol. 102: 1463-73 (1986)).
Such isoform diversity may be critical to maintain a specific pattern of protein distribution in neurons and polarized epithelial cells such as those of the kidney tubules that directionally transport ions and nutrients. It is likely that such tissues harbor additional isoforms of ankyrin. Antibodies raised against Ank.sub.R recognize a 210 kDa product in erythrocytes, and a 190 kDa polypeptide in kidney tissue (Davis et al., 1989; Morrow et al., 1991). The Ank.sub.R from both sources binds spectrin and Na, K-ATPase (Devarajan et al., 1994). However, Ank.sub.R -deficient NB/NB mice express the 190 kDa renal ankyrin, indicating that it is encoded by a gene distinct from ANK1, presumably an ANK3 gene (Bennett, 1992; Peters et al., 1993). Antibodies to Ank.sub.B cross-react with a 220 kDa peptide in kidney tissue, but Ank.sub.B cDNA probes hybridize only weakly to renal RNA (Otto et al., 1991), suggesting that the kidney Ank.sub.B peptide may also be the product of a distinct gene. Finally, antibodies to Ank.sub.G recognize polypeptides in the 190-72 kDa range in rat kidney (Kordeli et al., 1995), none of which have been further characterized.
After this work was first submitted, a report describing sequences Ank.sub.G in brain appeared (Kordeli et al., 1995). Comparing Ank.sub.G119 with the Ank.sub.G observed in brain reveals the ankyrin cloned to be an alternative transcript of the larger form identified in brain. We therefore now term this form Ank.sub.G119, rather than Ank.sub.K as previously reported in abstract form (Devarajan et al., 1995).
Ank.sub.G119 possesses only part of the repeats domain characteristic of all previously described ankyrins, and also deletes almost the entire regulatory domain. Ank.sub.G119 is expressed in kidney, placenta, and skeletal muscle, in Madin-Darby Canine Kidney (MDCK) cells (a collecting tubule line) and in cultured porcine proximal tubule cells (LLC-PK1). It is also present in low abundance in other cell types. The distribution of this small ankyrin in rat kidney cells and in confluent MDCK and LLC-PK1 cells is cytoplasmic and Golgi associated, unlike the plasma membrane localization of all other previously described ankyrin isoforms. Ank.sub.G119 also specifically binds MDCK cell .beta.I.SIGMA.* spectrin (erythroid like) in vitro with nanomolar affinity, and is coincidentally distributed in the Golgi apparatus with a previously described Golgi associated .beta.I.SIGMA.* spectrin (Beck et al., 1994). These findings significantly extend the known diversity of the ankyrins, confirm the presence of a Golgi associated .beta.I.SIGMA.* spectrin, and strongly imply that these two proteins function together as an integral part of the Golgi apparatus.